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Introduction

During the last 10 years an increasing number of water wells have been polluted by
pesticides or its break down products. BAM is among the most frequent found pesticide
residues in European groundwater. As pesticide analysis of drinking water is currently
being done by laboratory analysis, an in-line sensor will therefore be beneficial
for water quality monitoring. Cantilever-based assays for pesticide detection has
been reported [1,2], but few description of repeated measurements using cantilever-based detection systems
are available. As a central principle of a possible cantilever-based competitive assay,
we have tested the binding of a BAM antibody to a cantilever surface passive coated
with a BAM ovalbumine conjugate. In a working assay, the BAM molecules in a water
sample would compete with BAM attached to a cantilever surface for the binding to
anti-BAM monoclonal antibodies, similar to a BAM ELISA described by Bruun et al [3]. The binding of anti-BAM antibodies to the surface of the cantilever will change
the surface stress, causing bending of the cantilever. The bending is then detected
by a change in resistance of the imbedded piezoelectric layer in the cantilever [4-6]. To investigate whether the system is suited as a transducer for a pesticide bio-assay,
the variance of the cantilever bending signal during 10 antibody binding experiments
was analyzed. The mechanical properties of the cantilevers were also monitored by
measuring the cantilever bending profile, cantilever mass/stiffness, and antibody
fluorescent signal. This was repeated on the clean cantilevers, after the cantilevers
were functionalization with antigens, and after the antibody was added.

Figure 1.Experimental setup overview. (Above) A schematic overview of the fluidic setup; (Below) Flowchart of the BAM assay
on the CantiChip4® system .

In order to verify the binding of antibodies to the cantilever surface and control
for unspecific binding, a set of fluorescent pictures of Cy5 and Cy3 signal were taken
after spotting and antibody attachment. An optical surface profilometer (Polytech
TMS-100), based on light interference, was used to analyze the absolute bending of
the cantilevers on five experiments. To analyze the mass/stiffness values, a laser-based
vibrometer with a piezo actuator (Doppler Vibrometer Polytech MSA 500) was used on
eight experiments [8]. All chemicals used in the assay were purchased via Sigma Aldrich Denmark; only new
glassware was used and rinsed in Milli-Q water to avoid any unwanted effect from surfactants.

Results and discussion

Twenty chips were selected for the BAM assay based on signal stability while running
in air mode and a buffer flow. Of 20 experiments, only ten gave a signal when adding
BAM antibody (with five experiments giving a differential signal above 0.01 mV) (Figure
2). Seven experiments gave no differential signal, and three chips were discarded after
functionalization, due to too high initial voltage difference between the cantilevers.
A signal from the addition of specific BAM antibody, as well as from the addition
of unspecific antibody appeared on all 10 successful experiments. The differential
signals show a very diverse and distinct signal profile in between experiments, but
has a similar signal profile between the specific and the unspecific antibody on each
experiment (Figure 2).

Figure 2.Comparison of bending signals from 10 experiments. The differential signal between the two signal BAM-coated and two reference ovalbumine-coated
cantilevers is shown. Plotted as signal (mV) of (B + C) - (A + D) as a function of
time (s) during the addition of BAM antibody (left) and unspecific antibody (right).

Baseline noise was typically in the range of 0.004 to 0.002 mV. (Figure 3, left). As the absolute bending signal were not suited to evaluate the experiment,
the differential values of A(signal) - B(reference), C(reference) - D(signal), B(signal)
- C(signal), and A(reference) - D(reference) were plotted, a signal example from chip
117 is seen in Figure 3.

The deflection values showed a clear bending of all cantilevers after the functionalization
step (Figure 5, right). This was probably caused by salt deposits from the PBS buffer used in the
micro-spotting of BAM-ovalbumine conjugate and ovalbumine. The cantilevers returned
to their initial state after the experiment, probably caused by the removal of these
deposited salts from the functionalization step. A large variation on the resonance
frequency could explain the diverse signal variations obtained. The cantilevers showed
a slight increase in variation of the resonance frequency after the functionalization
step; but no significant difference could be seen after the experiment was performed
(Figure 5, left).

Figure 5.Cantilever resonance frequency and bending. (Left) Mass/stiffness ratios of each of the four cantilevers divided in three groups:
clean chip, after functionalization by micro-spotting, and after the addition of BAM
antibody . The values are an average of eight experiments (104, 108, 112, 116, 117,
118, 119, and 120). (Right) Average bending values (μm) of cantilever tip relative
to the chip body surface. Values are averages obtained from five experiments (116,
117, 118, 119, and 120).

Although three chips were discarded during the 20 experiments, the Cantion chips were
able to perform a continuous voltage readout lasting several days. The Cantion chips
could also be re-used following a rinsing protocol. This opens up the possibility
of regeneration of the surface chemistry by repeated assays, using only one sensor
in an automated system. However, the system was not found suitable as a platform for
a pesticide bio-assay in its current form, as the quality of the differential signal
was not repeatable. The fluorescent pictures of anti-BAM showed repeated attachment
only to the BAM functionalized cantilever surfaces, and no binding of unspecific Cy5
marked antibody. The signal variation is therefore unlikely to be caused only by variations
in the cantilever functionalization step. The variations are more likely caused by
minute changes in buffer pH, temperature, and salinity, as this affects the electromagnetic
field surrounding the cantilever (caused by the 2.5 V tension in the cantilever piezo
layer). The very large antibody concentrations needed to obtain a differential signal
on the system is believed to be the cause of the signal from the unspecific antibody
as it interacted with the cantilever surface, but this could not be proved in the
experiments.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

MHJ conceived the study. RT assisted in the design of the study. JA provided Anti-BAM
antibody and BAM hapten EQ0031. SS performed the cantilever bending and resonance
frequency measurements. MB performed the experiments, designed the study and wrote
the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This study is financed by the Danish consortium SENSOWAQ in collaboration with GEUS
(Grant no. 2104-06-0006 from the Danish Council for Strategic Research).